Effects of Shear on Specific Adhesion Receptor Expression and Binding in the Bact

描述(由申请人提供)：细菌通过受体对生物(宿主组织)和合成底物(例如，生物医学设备)的特异性黏附：配体与被吸附分子(例如，血浆蛋白质、碳水化合物和糖共轭)的相互作用是导致生物膜形成、宿主组织入侵、毒力和感染以及潜在死亡的一系列过程中的关键第一步。医院感染是美国第四大死因，每年有200万例(约占美国住院患者的10%)。在所有此类感染中，约有60%-70%与植入的医疗设备有关，2002年造成45亿美元的医疗费用，每年造成约99000人死亡。生物膜形成的第一步之一是细菌细胞通过“粘附素”(或受体)与存在于目标表面的配体分子的特异性粘连。防止这种最初的黏附结合可能会潜在地消除生物膜的形成和任何随后的感染。开发小分子疗法或疫苗以防止与生物医学设备的特定黏附，或者相反，制造旨在促进黏附和识别特定病原体的“芯片实验室”阵列，将需要在相关的流体动力学条件下关于细菌特定结合事件的详细信息。在体外鉴定细菌受体及其固定化配体上的结合表位的尝试是复杂的，(1)不能以定义和一致的方向呈现配体，(2)缺乏黏附受体表达的详细动力学，作为细胞生长和环境水动力学条件的函数，我们根据先前的工作假设，精确地量化细菌的特异性黏附将需要(1)控制结合配体的取向和表面密度；(2)确定细菌黏附受体的数量、亲和力和亲和力；以及(3)在适当的生长和流体剪切条件下，受体：配体对-ALL的结合作用的表征。我们的最终目标是开发一种通用的方案，定义细菌黏附受体：作为流体剪切函数的天然状态的配体相互作用-用于纯培养和混合培养的生物膜。与公共卫生相关：我们的最终目标是开发一种通用的方案，该方案将定义细菌黏附受体：作为流体剪切函数的天然状态下的配体相互作用-用于纯培养和混合培养的生物膜。在NIH的支持下，我们将使用表皮葡萄球菌(SE)与固定化纤维连接蛋白(FN)结合的模型纯培养系统来开发这一方案。我们在这个为期两年的项目中的具体目标是：1.作为主流流体剪切的函数，量化SE菌株通过纤维连接蛋白结合受体(FNBR)以受控取向和已知表面密度固定的FN的特异性结合。2.定量FNBR黏附素受体在SE表面表达的动力学，作为细菌生长条件和主流流体剪切力的函数；作为浮游细胞和贴壁细胞。

DESCRIPTION (provided by applicant): Bacterial specific adhesion to biological (host tissue) and synthetic substrata (e.g., biomedical devices) through receptor:ligand interactions with adsorbed molecules (e.g., blood plasma proteins, carbohydrates, and glycoconjugates) is a critical first step in a cascade of processes leading to biofilm formation; host tissue invasion, virulence and infection; and potentially death. Nosocomial infections are the fourth leading cause of death in the U.S. with >2 million cases annually (or ~10% of American hospital patients). About 60-70% of all such infections are associated with an implanted medical device causing >$4.5 billion medical costs in 2002 and ~99,000 deaths annually. One of the first steps in biofilm formation is the specific adhesion of bacterial cells via `adhesins' (or receptors) to ligand molecules present on the target surface. Prevention of this initial adherence binding could potentially abrogate biofilm formation and any subsequent infection. Development of small molecule therapies or vaccines to prevent specific adhesion to biomedical devices or, conversely, the fabrication of "lab-on-a-chip" arrays designed to promote adhesion and identify specific pathogens will require detailed information on bacterial specific binding events, under pertinent hydrodynamic conditions. Attempts to identify binding epitopes on both bacterial receptors and their immobilized ligands, in vitro, are complicated by (1) the inability to "present" the ligand in a defined and consistent orientation and (2) lack of detailed kinetics for adhesion receptor expression, as a function of cell growth and ambient hydrodynamic conditions, Based on prior work, we hypothesize that to precisely quantify bacterial specific adhesion will require (1) controlling the orientation and surface density of the binding ligand; (2) determination of the number, affinity and avidity of bacterial adhesion receptors; and (3) characterization of the binding interactions of the receptor:ligand pair - all under pertinent conditions of growth and fluid shear. Our ultimate goal is to develop a general protocol that will define bacterial adhesion receptor:ligand interactions, in their native states as a function of fluid shear - for both pure and mixed culture biofilms. PUBLIC HEALTH RELEVANCE: Our ultimate goal is to develop a general protocol that will define bacterial adhesion receptor:ligand interactions, in their native states as a function of fluid shear - for both pure and mixed culture biofilms. With NIH support, we will develop this protocol using the model pure culture system of Staphylococcus epidermidis (SE) binding to immobilized fibronectin (FN). Our specific aims in this two-year project will be: 1. Quantify, as a function of prevailing fluid shear, the specific binding of SE strains via fibronectin binding receptors (FNBR) to FN immobilized in a controlled orientation and known surface density. 2. Quantify the kinetics of FNBR adhesin receptor expression on SE surfaces, as function of bacterial growth condition and prevailing fluid shear; both as planktonic and adherent cells.